Strain of micro-organism lactobacillus fermentum me-3 as novel anti-microbial and anti-oxidative probiotic

ABSTRACT

The strain of micro-organism  Lactobacillus fermentum  ME-3 is a novel anti-microbial and anti-oxidative probiotic. It has a high anti-microbial effect on  Escherichia coli, Shigella sonnei, Staphylococcus aureus, Salmonella typhimurium,  and moderate activity against  Helicobacter pylori  strains. The strain of micro-organism possesses Mn-superoxide dismutase and both its lysates and intact cells have high anti-oxidative activity, increasing the glutathione red-ox ratio in blood sera and able to capture toxic hydroxyl radicals. The strain of micro-organism could be used as a probiotic for the production of functional food (yoghurt, cheese) and non-comestibles (tablets, capsules) for the prophylaxis of intestinal and uroinfections, both for the prevention and treatment of chronic diseases, caused by prolonged oxidative stress.

TECHNICAL FIELD

[0001] The present invention relates to biotechnology and will be usedas a novel probiotic applied in the production of functional food(yoghurt, cheese) and non-food preparations (tablets, capsules) for theprevention or treatment of different diseases.

BACKGROUND ART

[0002] Probiotics are living microbial food additives, that havebeneficial effect on the microbiological balance of the intestine andhuman health. Probiotics are used as functional food. Functional food isfoodstuff, consumed additionally to usual food and containingbio-preparations (incl. probiotics) or other components favourablyinfluencing human health or decreasing disease risks.

[0003] Probiotics are consumed as components of food (probiotic yoghurtor cheese) or non-food preparations (lyophilised microbial cultures).

[0004] Most of probiotics are lactic acid bacteria, mainly lactobacilli.Lactobacilli are non-pathogenic micro-organisms, colonising the humanintestinal and urogenital tract from early childhood to old age.Nowadays, several commercial probiotic lactobacilli are successfullyused, among which Lactobacillus rhamnosus GG (Saxelin M. LactobacillusGG—a human probiotic strain with thorough clinical documentation. FoodRev Int 1997; 13:293-313) is the best known. Recently some new strainsof lactobacilli have been described and patented, for example L.reuterii (Korea patent KR211529, C12N 1/20, Korea Institute ScienceTechnology, 1999), isolated from animal organism and for this reasoninappropriate for human usage.

[0005] Several strains of Lactobacillus fermentum are used forcorrection and stabilisation of intestinal micro-flora in case ofdysbacterioses and urogenital infections with different ethiologies. Thestrain of micro-organism Lactobacillus fermentum 39 is used forproducing the bacterial biological preparation (PCT/SU89/00264 (WO91/05852), C12N 1/20, A61K 35/74, University of Tartu, 1991). The strainLactobacillus fermentum 90-TS-4 (RU2133272, C12N 1/20, A61K 35/74, AkivoLentsner et al., 1999) is characterised by lectin typing as amannose-sensitive profile of the cell wall. The preparation isprescribed for use in gynaecology.

[0006] There are some well-known probiotics, targeted against only onepathogen (for example Salmonella) (U.S. Pat. No. 5,478,557, A61K 35/74,US Agriculture, 1995; U.S. Pat. No. 5,340,577, A61K 35/74, US Army,1994). Up to the present no strain of lactobacilli with an extensiveanti-microbial effect against numerous pathogens and opportunisticpathogens has been described.

[0007] Likewise, yet no such strain of micro-organism is known thatcould have natural antibiotic resistance against drugs most frequentlyused in the treatment of infections. This property would permit to usesuch strains in case of antibiotic-treated patients. A set of differentmicro-organisms is used in veterinary, containing also one strain of L.fermentum, but this strain does not have a concurrent anti-microbial andanti-oxidative effect (RU2119796, A61K 35/66, Zakr{overscore (o)}tojeaktsionernoje obshestvo “BAKS”, 1998).

[0008] Anti-oxidative preparations like vitamin E and C, betacarotenea.o. nowadays get much attention in connection to healthy nutrition.Excessive formation of reactive oxygen species (ROS) in tissuerespiration can cause the damage of cells and the course of tissues. Theformation of active oxygen may depend on some stress factors, such asalcohol, peroxides, and some drugs.

[0009] Usually excessive oxidation is closely connected with nutritionaldiseases, age, arteriosclerosis, misfunctions of the central nervoussystem and the intestinal tract, cancer a.o. pathological conditions. Anorganism has several defence systems against the toxigenicity of oxygen.It is important to take anti-oxidative substances to guarantee thefunctioning of these systems.

[0010] Of known solutions, the closest to this invention is the patentdescribing anti-oxidative food, an anti-oxidative preparation and amethod of antioxidation (EPO649603, A23L 3/3472, A23L 3/3571, OtsukaPharma Co Ltd. 1995). The object of this invention is a preparation thatcontains a natural substance involving mangan (leaves of tea plant) andthe micro-organism Lactobacillus plantarum that produces catalase and asuperoxidase-dismutase system, thus increasing the anti-oxidativeactivity of the host organism. The authors of this invention declarethat the preparation prevents diseases developing due to active oxygen.However, they do not describe the effect of particular Lactobacillusstrain with a decreasing anti-oxidative activity or capturing hydroxylradicals in vitro. Besides that, this strain of micro-organism is alsoimperfect because for getting the presumable anti-oxidative effect in anorganism (in vivo), it is necessary to add some Mn-containing rawmaterial (leaves of tea plant) to the preparation, because it is only inthis case its SOD (superoxide dismutase) activity is realised.

DISCLOSURE OF THE INVENTION

[0011] The aim of this invention is to present the strain ofmicro-organism as a novel anti-microbial and anti-oxidative probioticfor use in pharmaceutical and food industry, also in medicine as anantibiotic-resistant preparation for the prophylaxis and treatment ofgastrointestinal and uroinfections, and against oxidative stress.

[0012] The object of the investigation—the strain of microorganismLactobacillus fermentum ME-3 was isolated from a faecal sample of ahealthy child during a comparative study of the micro-flora of Estonianand Swedish children, using MRS (Oxoid) media and cultivating it in aCO₂ environment (Sepp et al., Intestinal microflora of Estonian andSwedish infants, Acta Paediatrica, 1997, 86, 956-961).

[0013] The strain of micro-organism Lactobacillus fermentum ME-3 wasisolated by seeding the dilutions of the faeces of healthy one-year-oldEstonian child (10⁻²-10⁻⁷ in phosphate buffer with 0.04% thioglycolacid; pH 7.2). The dilutions were seeded on freshly prepared MRSagar-media and cultivated at 37° C. in a CO₂ environment. The strain,which is the object of invention, was isolated from a 10⁻⁵ dilution onthe basis of the characteristic morphology of colonies and cells. Aprovisional and more precise identification followed as described next.Using additional tests, the strain was selected from other lactobacilliisolated from the same child on the basis of its special properties.

[0014] The fact that the microbial strain Lactobacillus fermentum ME-3originates from the intestinal tract of a healthy child proves its GRAS(generally recognised as safe) status, i.e. that this strain ofmicro-organism is harmless for human organism and it is suitable fororal application.

[0015] Cultural-morphological characteristics were determined aftercultivating the strain on MRS agar and in MRS broth media (OXOID).Microbial cells are Gram-positive rods of regular shape located inparallel chains, nonspore, of medium thickness and different length(2×3-5 μm).

[0016] Physiological-biochemical characteristics: MRS broth was suitablefor cultivating the microbial strain during 24-48 hours in a 10% CO₂environment, after which homogeneous turbid growth occurred in thebroth. The colonies of micro-organism on MRS agar are white, rounded,with a regular edging.

[0017] The optimal growth temperature is 37° C., it multiplies also at45° C., but it does not grow at 15° C. The optimal growth environment isat pH 6.5.

[0018] The negative catalase test, gas production by fermentation ofglucose, production of NH₃ from arginine, and lysozyme production arethe main properties. During reproduction in milk it produces 1.07% ofacid.

[0019] The strain with above-mentioned characteristics was identified onthe basis of biochemical activity with API 50 CHL System (BioMerieux,France) kit as Lactobacillus fermentum (ID% 99.6, T 0.87, only 1 testcontra). The following sugars and alcohols were fermented—ribose,galactose, D-glucose, D-fructose, D-mannose, esculine, maltose, lactose,melibiose, saccharose, D-raffinose, D-tagatose and gluconate.

[0020] The profile of the metabolites of Lactobacillus fermentum ME-3was characteristic of heterofermentative metabolism, determinated by thegas chromatographic method (Hewlett-Packard model 6890). The profile offermentation depended on environment of incubation: besides lactic andacetic acids a big amount of succinic acid was produced in a CO₂environment, but in an anaerobic environment much of ethanol wasproduced in addition to the above-mentioned substances (Table 1). Bothsuccinic acid and ethanol can strengthen the stable properties of themicrobial strain in milk fermented by this strain. TABLE 1 Theconcentration of acetic acid, lactic acid, succinic acid and ethanol(mg/ml) in MRS media in cultivation of Lactobacillus fermentum ME-3 inmicroaerophilic and anaerobic environment during 24 and 48 h.Lactobacillus Lactic Acetic Succinic fermentum acid acid acid EthanolME-3 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h CO₂ 10.6 11.1 0.8 0.9 18.419.5 9.8 7.5 environment Anaerobic 8.2 8.8 1.0 1.0 5.7 9.7 7 33.3environment

[0021] Molecular Identification.

[0022] Molecular identification by ITS-PCR (internal transcribedspacer-polymerase chain reaction) using Lactobacillus fermentum ATCC14931 as the reference strain verified the previous identification withAPI 50 CHL.

[0023] The micro-organism with the above-mentioned properties wasdeposited in Deutsche Sammlung für Mikroorganismen und ZellkulturenGmbH-s, the registration number of the deposite is DSM 14241(19.04.2001).

[0024] Anti-Microbial Activity

[0025]Lactobacillus fermentum ME-3 expresses a high anti-microbialeffect on Escherichia coli, Shigella sonnei, Staphylococcus aureus,Salmonella typhimurium 1 and 2, and Helicobacter pylori strains in vitro(Table 2). TABLE 2 Anti-microbial activity of strain Lactobacillusfermentum ME-3 on modified MRS-agar, in MRS broth and milk.Lactobacillus Salmonella fermentum Escherichia Shigella Staphylococcustyphimurium Helicobacter ME-3 coli sonnei aureus 1 and 2 pylori MRS-agarInhibition zone (mm) CO₂/ 24/22 26/21 20/19 25.8/ 23.8/ 13.2/13.1anaerobic 24.7 19.7 environment MRS broth Decrease of total count(log₁₀) compared with initial count log 6.0 Log log 0.8 log log not 6.76.3 3.8 determined Milk Suppression after different interval of time(24-48 h) 24 t 32 t 24 t 32 t 48 t not determined

[0026] Using milk fermentation it was possible to show that pathogensinoculated into milk were killed in 24-48 h if milk was fermented withLactobacillus fermentum ME-3. Such property of the strain could help toprevent the multiplication of pathogens in products (yoghurt, cheese)fermented by this strain, and prevent food infections. Organic acids andethanol produced by Lactobacillus fermentum ME-3 could ensure the highanti-microbial effect of this microbe.

[0027] Resistance to Antibiotics

[0028] According to a disk-diffusion test (BBL Sensi disks) and anE-test (AB Biodisk, Solna) Lactobacillus fermentum ME-3 was resistant tometronidazole, ofloxacin, aztreonam, cefoxitin and TMP-SMX. This allowsto use the strain L. fermentum ME-3 as a preparation accompanyingantibiotic treatment in case of intestinal and uroinfections.

[0029] Surface Structures of Microbial Cell

[0030] The carbohydrate profile of the surface structure of microbialcells of Lactobacillus fermentum ME-3 was determined by lectin typing.The strain of lactobacilli agglutinated with Griffonia simplifolia Ilectin, which is specific to Gal and GalNAc ligands in the cell wall.

[0031] The strain Lactobacillus fermentum ME-3 did not react with thefollowing other lectins: Concanavalin ensiformis (Con A), Griffoniasimplicifolia II, Arachis hypogaea (PNA), Vicia sativa (VSA) and Tritiumvulgaris (WGA).

[0032] Hence the special composition of the glycocalyx of the cell wallof Lactobacillus ME-3 became clear with lectin typing, it containedresidues of galactose and N-acetyl-galactose-amine. These compounds actas adhesins for engaging the receptors of mucosa on the epithelial cellsof the upper urinary tract.

[0033] This is a possibility for blocking the mannose-resistant pili ofEscherichia coli that makes our strain applicable in the prophylaxis ofurinary tract infections.

[0034] Anti-Oxidative Properties

[0035] Lactobacilli were incubated in a MRS broth (Oxoid Ltd.) for 24 hand centrifuged at 4° C. (1500 p/min) 10 min for getting a precipitate,washed with isotonic salt (4° C.) and suspended to the density of 1.15%KCl (Sigma, USA). The density of the suspension was at OD₆₀₀ 1.1 10⁹bacterial cells in ml⁻¹). To get lysates, the cells were disrupted bysonification (B-12 Branson Sonic Power Company, Danbury, Conn.) in 35vibrations s⁻¹ 10 min in an ice bath and then for 10 min at −18° C. Thesuspension was centrifuged at 4° C. 10000 g/r for 10 min and thesupernatant was filtered (MILLEY-GS, sterile, 0.22 μm; Millipore S. A.,67 Molsheim, France) to get a cell-free extract. Lactobacillus fermentumME-3 cells and lysate produced H₂O₂ in a remarkable amount (Table 3).TABLE 3 Total anti-oxidative capacity of Lactobacillus fermentum strainsME-3 and E-338-1-1 (according to LA and TAS tests), hydrogen peroxidecontent, glutathione red-ox ratio and activity of superoxide dismutase.Lactobacillus Lactobacillus Properties fermentum ME-3 fermentumE-338-1-1 Intact cells Intact cells TAA in LA-test   29 ± 0.7 (n = 5) 0(%) TAS (mmol/L)  0.16 ± 0.03 (n = 5) 0 H₂O₂ (μg/ml)   31 ± 26 (n = 3) 49 ± 20 (n = 3) Lysate of cells Lysate of cells LA-test (%)   59 ± 3.8(n = 5) 0 H₂O₂ (μg/ml)   229 ± 37 (n = 4) 137 ± 25 (n = 3) TGSH  12.5 ±4.1  5.5 ± 3.0 GSSG (μg/ml)  2.59 ± 2.01  5.5 ± 2.4 GSH (μg/ml)  9.95 ±3.30 Marks GSSG/GSH  0.28 ± 0.17 0^(e) SOD (U/mg 0.859 ± 0.309 (n = 3)Not determined protein)

[0036]Lactobacillus fermentum ME-3 has a Mn-SOD activity determined byelectrophoresis. For determining the SOD type L. fermentum ME-3cell-free extract (30 μg protein) was separated on 10% not-denaturatedpolyamide-acrylic gel. SOD isoenzyme was determined by influencing thisgel with 15 mM H₂O₂, after which the SOD activity persisted.Explanation: H₂O₂ inhibits Fe-SOD, but does not inhibit Mn-SOD. Thisproves that Lactobacillus fermentum ME-3 has Mn-SOD activity.

[0037] The strain Lactobacillus fermentum ME-3 showes a high TAA (totalantioxidative activity) value in a lipid environment on the basis of alinolenic acid test, also a high TAS (total antioxidative status) valuein a hydrate environment (Randox kit, UK). In Table 3, data of theanti-oxidative strain Lactobacillus fermentum E-338-1 is added forcomparison (Table 3).

[0038] The cells and lysates of the strain Lactobacillus fermentum ME-3catch hydroxyl radicals, this has been proven by the terephthalic acidmethod (27%±5%). 15 mM of reduced glutathione was used for comparison asa well-known scavenger of hydroxyl radicals (84±4.6%). Lactobacillusfermentum ME-3 survived in a highly oxidative H₂O₂ environment.

[0039] Re-cultivation of the lyophilised culture kept in roomtemperature for a long time proved the viability of the strain and thepersistence of properties. This ensures that the lyophilised strain ofLactobacillus fermentum ME-3 could be used as a non-comestible productin a scheme of functional food.

BEST MODE FOR CARRYING OUT THE INVENTION

[0040] An example of the preparation of a yoghurt with highlyanti-oxidative properties based on the strain Lactobacillus fermentumME-3 and the trial of consuming the yoghurt by healthy volunteers.

[0041]Lactobacillus fermentum ME-3 pure culture in 0.15% MRS-agar isused for producing the yoghurt, additionally the pure cultures ofLactobacillus plantarum and Lactobacillus buchneri are seeded into freshgoat milk autoclaved for 20 min at 110° C. Three cultures of thesestrains of lactobacilli are mixed in equal proportions together with 2%of Streptococcus thermophilus and are added in 0.2% of content intoautoclaved goat milk.

[0042]Lactobacillus fermentum ME-3 with strains of lactobacilli andstreptococci will guarantee tasty and highly anti-oxidative yoghurt(Table 4). TABLE 4 The anti-oxidative activity of Lactobacillusfermentum ME-3 pure culture and probiotic yoghurt Total anti-oxidativeactivity (TAA %) Strain Cells Yoghurt Lactobacillus 29 70 fermentum ME-3

[0043] In tables 5 and 6, the changes of the intestinal micro-flora andindices of oxidative stress of blood sera of healthy volunteers areshown before and after taking the probiotic goat milk yoghurt during 3weeks. These changes prove the anti-oxidative (incl. anti-atherogenic)effect on human organism.

[0044] Even a higher total anti-oxidative activity of goat milk yoghurtcompared with the total anti-oxidative activity of intact microbialcells of Lactobacillus fermentum ME-3 is shown in table 5.

[0045] Additive microbial strains ensure the standard acidity andconsistence of yoghurt. TABLE 5 The changes of intestinal micro-flora ofhealthy volunteers (n = 16) before and after consuming probiotic goatmilk yoghurt during 3 weeks Before After Lactoflora Persons LactofloraPersons colonised ratio (%) colonised ratio (%) Consuming goat milkyoghurt (n = 16 persons) L. fermentum  4* 0.7-5.77  16* 0.5-49.9# Takinggoat milk (n = 4 persons) L. fermentum 0 0  1   0-32.9

[0046] TABLE 6 The indices of oxidative stress of blood sera ofvolunteers (n = 16) before and after consuming probiotic goat milkyoghurt during 3 weeks Standard Blood sera Blood trial Properties degreebefore trial after trial Increase TAA (LA-test,   36 ± 4.5   38 ± 3.5  45 ± 3.4 16% %) TAS, mmol/L  1.2 ± 0.2 0.82 ± 0.14 1.14 ± 0.08 29%Glutathione 0.17 ± 0.08 0.15 ± 0.01 0.11 ± 0.035 −32% red-ox ratio(GSSG/GSH) LDL lag-phase >30 min   41 ± 7.9   46 ± 8.6 11% (time ofresistance) Basic value of  <0.3 0.27 ± 0.06 0.23 ± 0.06 −15% dieneconjugates (value of extinction) Ox LDL (U/L) >127   98 ± 12   81 ± 19−18%

[0047] Thus all parameters determined in the blood sera of healthyvolunteers changed beneficially during the 3-week yoghurt trial.

[0048] The application of the invention is not limited to theabove-described example of achievement. In the range of the patentclaim, some other variants of use are possible, for example theproduction of probiotic cheese and other milk products.

[0049] Statement

[0050] The aim of present invention is to offer a strain ofmicroorganism as a novel anti-microbial and anti-oxidative probiotic foruse in pharmaceutical and food industry, also in medicine as anantibiotic resistant preparation for prophylaxis and treatment ofgastrointestinal and uroinfections, also against oxidative stress (P.3,line 33-35, P.4, line 1-3)

[0051] Concerning the antimicrobial activity (bacteriostatic influence)of the object of invention, the strain Lactobacillus fermentum ME-3expresses anti-microbial effect beside others on Shigella sonnei,Salmonella typhimurium ja Helicobacter pylori strains (Page 6, line21-25; Page 7, Table 2). In addition, the property of Lactobacillusfermentum ME-3 to kill the food borne pathogens in milk (bacteriocidiceffect) is firstly described (Page 7, Table 2 and line 6-11).

[0052] The innate resistance of Lactobacillus fermentum ME-3 againstantimicrobial preparations (TMP-SMX, ofloxacin, aztreonam, cefoxitin andmetronidazole) allows to use it as a preparation accompanying antibiotictreatment in case of gastrointestinal and uroinfections (Page 7, line17-19; Page 8, line 1-3). This property has not been described elsewherebefore.

[0053] The unique carbohydrate profile of the cell wall of Lactobacillusfermentum ME-3 enables to prevent the adhesion of uropathogenicEscherichia coli to the epithelial cells of the upper urinary tract, aproperty that makes our strain applicable in the prophylaxis of urinarytract infections (Page 8, line 5-26)and has never been described before.

[0054] Concerning the antioxidative activity of the strain Lactobacillusfermentum ME-3 as the object of the present invention the differentspecific, principal and novel parameters were firstly described likeexpression of MnSOD, high-grade total antioxidative status (TAS,verified by internationally accepted method), principal parametres ofglutathione (a signal molecule and central cellular antioxidant) systemand the value of glutathione redox ratio (Page, 9 Table 3; Page 10, line1-11, 15-19, 23-26).

[0055] Any antioxidativity (including antiatherogenicity) parametersfound in human trials (in vivo trials) were not made public elsewhere.Therefore, only in this invention, an influence of consumption of ME-3on human blood sera specific indices was described (Page 13-14, Table 6)and disclosed the appropriate numerical values. Actually, consideringmainly these parameters (significant increase of TAS and oxygenresistance of LDL, lowering the level of oxidized LDL and its dieneconjugates altogether indicate improvement of systemic antioxidativityand also significant lowering of cellular oxidative stress) it can beclaimed that strain Lactobacillus fermentum ME-3 is a novelantioxidative (anti-atherogenic) probiotic (Page 11, line 25-30).

[0056] The persistence of the novel strain in gastrointestinal tractafter consumption and the beneficial influence on the composition of theintestinal lactobacilli are desribed for the first time (Page 12, Table5, line 14-22).

[0057] Thus the strain of microorganism Lactobacillus fermentum ME-3(DSM14241) figures a novel antimirobial and antioxidative (anti-atherogenic)probiotic for use in pharmacy and food industry, and in medicine as apreparation resistant to some antimicrobials useful for the prophylaxisand as a preparation accompanying antibiotic therapy of gastrointestinaland urinary tract infections, also against chronic diseases (incl.atherosclerosis) induced by prolonged high-grade oxidative stress.

1. The strain of micro-organism Lactobacillus fermentum ME-3 DSM 14241as a novel anti-microbial and anti-oxidative probiotic for use inpharmacy and food industry and in medicine as a preparation resistant toantibiotics for the prophylaxis and treatment of intestinal anduroinfections, also against oxidative stress.